Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.

Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked ß-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.

Factors Predicting Loss of Remission in Crohn's Disease Patients in Endoscopic Remission in the Real World: Results From TARGET-IBD.

BACKGROUND: There is limited evidence that histologic remission improves outcomes in Crohn's disease (CD). We aimed to characterize a cohort of patients with CD in endoscopic remission and explore factors associated with subsequent loss of remission (LOR). METHODS: In total, 4474 patients were enrolled in TARGET-IBD, a longitudinal, observational cohort study. Patients with a normal steroid-free colonoscopy (index) were defined as "in endoscopic remission" and were followed for LOR, defined as presence of inflammation, erosion, ulceration, or stricturing on a subsequent colonoscopy or commencement of steroids. Histologic activity was dichotomized using standard of care reports for active inflammation. Unadjusted and multivariable-adjusted Cox proportional hazards regression models were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of LOR in relation to independent variables. RESULTS: Of 658 patients with CD with steroid-free endoscopic remission, the majority were female (57%), white (83%), non-Hispanic (93%); 20% had ileal and 20% isolated colonic disease. Inflammatory (B1) disease was the most common phenotype (43%). Of these 658 patients, 257 (39%) had histologic inflammation on index colonoscopy. Histologic inflammation at index colonoscopy was associated with nearly twice the LOR risk (HR 1.96, 95% CI: 1.50-2.57) with median time to relapse of 1.20 years. Biologic use at index was associated with lower LOR risk (monotherapy, HR 0.61, 95% CI: 0.45-0.82; combination therapy, HR 0.43, 95% CI: 0.28-0.66). CONCLUSIONS: Active histologic inflammation despite endoscopic remission, and lack of biologic use were independently associated with risk of subsequent LOR, providing evidence that histologic remission may impart improved outcomes in patients with CD.

Improvements in Patient-Reported Outcomes After Treatment With Deucravacitinib in Patients With Psoriatic Arthritis: Results From a Randomized Phase 2 Trial.

OBJECTIVE: Deucravacitinib, a tyrosine kinase 2 inhibitor, was assessed in a phase 2 trial in patients with active psoriatic arthritis (PsA). Here, we report effects of deucravacitinib from the patient perspective. METHODS: This phase 2, double-blind trial (NCT03881059) randomized patients with active PsA 1:1:1 to deucravacitinib 6 mg once daily (QD), 12 mg QD, or placebo, for 16 weeks. Key secondary end points were changes from baseline (CFBs) at week 16 in Health Assessment Questionnaire-Disability Index (HAQ-DI) and 36-item Short-Form Health Survey (SF-36) physical component summary (PCS) scores. Additional patient-reported outcomes (PROs) assessed disease impact, including fatigue, pain, and mental health. The mean CFBs in PROs and percentages of patients reporting improvements with minimum clinically important differences (MCIDs) or scores of greater than normal values were also assessed. RESULTS: This study comprised 203 patients (51.2% female; mean ± SD age, 49.8 ± 13.5 years). At week 16, the adjusted mean difference (95% confidence interval) versus placebo in HAQ-DI and SF-36 PCS CFB was significant for each deucravacitinib group (HAQ-DI 6 mg, -0.26 [-0.42 to -0.10], P = 0.0020; HAQ-DI 12 mg, -0.28 [-0.45 to -0.12], P = 0.0008; SF-36 PCS 6 mg, 3.3 [0.9 to 5.7], P = 0.0062; SF-36 PCS 12 mg, 3.5 [1.1 to 5.9], P = 0.0042). MCID at week 16 were reported for all PROs with either dose of deucravacitinib. Improvements of MCID or to normative values were reported by more patients receiving deucravacitinib than placebo. CONCLUSION: Deucravacitinib groups demonstrate significant and clinically meaningful improvements in PROs versus placebo in patients with active PsA, which warrants further study.

Novel MFSD7-ATP5I fusion promotes migration and invasion of human sarcoma.

Fusion genes have been implicated in the development and progression of several types of sarcomas, serving as valuable diagnostic and prognostic markers, as well as potential therapeutic targets. We discovered a novel major facilitator superfamily domain-containing 7 (MFSD7) and adenosine triphosphate 5I (ATP5I) gene fusion from sarcomas. In this study, the MFSD7-ATP5I fusion transcript was screened using RNA sequencing in 55 sarcoma samples and sixteen normal samples. The MFSD7-ATP5I fusion transcript was detected in 58% of sarcoma samples. The correlation between the expression of MFSD7-ATP5I fusion transcript and clinicopathological information was analyzed, and MFSD7-ATP5I expression is associated with marked pleomorphism and lower tumor necrosis. Cell migration and invasion was significantly reduced by knock-down of MFSD7-ATP5I. Cell migration and invasion was increased by overexpression of MFSD7-ATP5I. A phosphokinase assay demonstrated that MFSD7-ATP5I is involved in the GSK-3 pathway. The current study found that MFSD7-ATP5I is associated with increasing pleomorphism and decreasing necrosis of tumors. And our gain and loss of function experiments prove that MFSD7-ATP5I promotes the invasiveness of tumor cells.

Enhanced Association of Tau Pathology and Cognitive Impairment in Mild Cognitive Impairment Subjects with Behavior Symptoms.

BACKGROUND: Mild cognitive impairment (MCI) individuals with neuropsychiatric symptoms (NPS) are more likely to develop dementia. OBJECTIVE: We sought to understand the relationship between neuroimaging markers such as tau pathology and cognitive symptoms both with and without the presence of NPS during the prodromal period of Alzheimer's disease. METHODS: A total of 151 MCI subjects with tau positron emission tomographic (PET) scanning with 18F AV-1451, amyloid-ß (Aß) PET scanning with florbetapir or florbetaben, magnetic resonance imaging, and cognitive and behavioral evaluations were selected from the Alzheimer's Disease Neuroimaging Initiative. A 4-group division approach was proposed using amyloid (A-/A+) and behavior (B-/B+) status: A-B-, A-B+, A+B-, and A+B+. Pearson's correlation test was conducted for each group to examine the association between tau deposition and cognitive performance. RESULTS: No statistically significant association between tau deposition and cognitive impairment was found for subjects without behavior symptoms in either the A-B-or A+B-groups after correction for false discovery rate. In contrast, tau deposition was found to be significantly associated with cognitive impairment in entorhinal cortex and temporal pole for the A-B+ group and nearly the whole cerebrum for the A+B+ group. CONCLUSION: Enhanced associations between tauopathy and cognitive impairment are present in MCI subjects with behavior symptoms, which is more prominent in the presence of elevated amyloid pathology. MCI individuals with NPS may thus be at greater risk for further cognitive decline with the increase of tau deposition in comparison to those without NPS.

Brain amyloid burden, sleep, and 24-hour rest/activity rhythms: screening findings from the Anti-Amyloid Treatment in Asymptomatic Alzheimer's and Longitudinal Evaluation of Amyloid Risk and Neurodegeneration Studies.

STUDY OBJECTIVES: To examine in a subsample at the screening phase of a clinical trial of a ß-amyloid (Aß) antibody whether disturbed sleep and altered 24-hour rest/activity rhythms (RARs) may serve as markers of preclinical Alzheimer's disease (AD). METHODS: Overall, 26 Aß-positive (Aß+) and 33 Aß-negative (Aß-) cognitively unimpaired participants (mean age = 71.3 ± 4.6 years, 59% women) from the Anti-Amyloid Treatment in Asymptomatic Alzheimer's (A4) and the Longitudinal Evaluation of Amyloid Risk and Neurodegeneration (LEARN) studies, respectively, wore actigraphs for 5.66 ± 0.88 24-hour periods. We computed standard sleep parameters, standard RAR metrics (mean estimating statistic of rhythm, amplitude, acrophase, interdaily stability, intradaily variability, relative amplitude), and performed a novel RAR analysis (function-on-scalar regression [FOSR]). RESULTS: We were unable to detect any differences between Aß+ and Aß- participants in standard sleep parameters or RAR metrics with our sample size. When we used novel FOSR methods, however, Aß+ participants had lower activity levels than Aß- participants in the late night through early morning (11:30 pm to 3:00 am), and higher levels in the early morning (4:30 am to 8:30 am) and from midday through late afternoon (12:30 pm to 5:30 pm; all p < .05). Aß+ participants also had higher variability in activity across days from 9:30 pm to 1:00 am and 4:30 am to 8:30 am, and lower variability from 2:30 am to 3:30 am (all p < .05). CONCLUSIONS: Although we found no association of preclinical AD with standard actigraphic sleep or RAR metrics, a novel data-driven analytic method identified temporally "local" RAR alterations in preclinical AD.

Disparities by Race and Ethnicity Among Adults Recruited for a Preclinical Alzheimer Disease Trial.

Importance: Underrepresentation of many racial/ethnic groups in Alzheimer disease (AD) clinical trials limits generalizability of results and hinders opportunities to examine potential effect modification of candidate treatments. Objective: To examine racial and ethnic differences in recruitment methods and trial eligibility in a multisite preclinical AD trial. Design, Setting, and Participants: This cross-sectional study analyzed screening data from the Anti-Amyloid in Asymptomatic AD study, collected from April 2014 to December 2017. Participants were categorized into 5 mutually exclusive ethnic/racial groups (ie, Hispanic, Black, White, Asian, and other) using participant self-report. Data were analyzed from May through December 2020 and included 5945 cognitively unimpaired older adults between the ages of 65 and 85 years screened at North American study sites. Main Outcomes and Measures: Primary outcomes included recruitment sources, study eligibility, and ineligibility reasons. To assess the probability of trial eligibility, regression analyses were performed for the likelihood of being eligible after the first screening visit involving clinical and cognitive assessments. Results: Screening data were included for 5945 participants at North American sites (mean [SD] age, 71.7 [4.9] years; 3524 women [59.3%]; 5107 White [85.9%], 323 Black [5.4%], 261 Hispanic [4.4%], 112 Asian [1.9%], and 142 [2.4%] who reported race or ethnicity as other). Recruitment sources differed by race and ethnicity. While White participants were recruited through a variety of sources, site local recruitment efforts resulted in the majority of Black (218 [69.2%]), Hispanic (154 [59.7%]), and Asian (61 [55.5%]) participants. Participants from underrepresented groups had lower mean years of education (eg, mean [SD] years: Hispanic participants, 15.5 [3.2] years vs White participants, 16.7 [2.8] years) and more frequently were women (226 [70.0%] Black participants vs 1364 [58.5%] White participants), were unmarried (184 [56.9%] Black participants vs 1364 [26.7%] White participants), and had nonspousal study partners (237 [73.4%] Black participants vs 2147 [42.0%] White participants). They were more frequently excluded for failure to meet cognitive inclusion criteria (eg, screen failures by specific inclusion criteria: 147 [45.5%] Black participants vs 1338 [26.2%] White participants). Compared with White participants, Black (odds ratio [OR], 0.43; 95% CI, 0.34-0.54; P < .001), Hispanic (OR, 0.53; 95% CI, 0.41-0.69; P < .001), and Asian participants (OR, 0.56; 95% CI, 0.38-0.82; P = .003) were less likely to be eligible after screening visit 1. Conclusions and Relevance: Racial/ethnic groups differed in sources of recruitment, reasons for screen failure, and overall probability of eligibility in a preclinical AD trial. These results highlight the need for improved recruitment strategies and careful consideration of eligibility criteria when planning preclinical AD clinical trials.

Real-world analysis of hospitalizations in patients with epilepsy and treated with perampanel.

OBJECTIVES: (1) To evaluate risk of hospitalization following initiation of perampanel (pre- and post-analysis) and (2) to compare hospitalization rates following initiation of perampanel vs lacosamide. METHODS: Patients were identified from Symphony Health's Patient Integrated Database if they had a prescription for perampanel (July 1, 2014-June 30, 2016). Patients 4-11 years of age with any partial-onset seizure (POS) or ≥12 years of age with any POS or primary generalized tonic-clonic seizure (GTCS) (pre-post); or ≥12 years of age (perampanel vs lacosamide). The first fill of perampanel ("index date") marked the start of the analysis period. Patients had ≥1 additional fill for perampanel and ≥2 diagnoses for epilepsy or nonfebrile convulsion diagnosis during pre-index (based on ICD-9/ICD-10 codes). Patients were matched using a 1:1 propensity scoring method for the perampanel vs lacosamide analysis. Primary outcome was hospitalization during the one year following medication initiation. RESULTS: Pre- and post-perampanel: N = 1771 (mean age 34 years, 55% female). One-year all-cause hospitalization risk ratio was 0.76 (P < .05) and 36.2% with hospitalization during the pre-period vs 29.5% in the follow-up. One-year epilepsy-related inpatient hospitalization risk ratio was 0.72 (P < .05) and 30.8% with hospitalization during the pre-period vs 23.9% during follow-up. In the perampanel and lacosamide cohorts, N = 1717 per cohort after matching, most baseline demographics were balanced. A higher percentage of subjects were prescribed ≥3 anti-seizure medications for perampanel vs lacosamide (60.5% vs 57.7%, P < .001). The perampanel cohort had a 9.6% reduction in all-cause hospitalizations vs 5.8% for the lacosamide cohort (P < .05). Epilepsy-related hospitalizations decreased from the pre-index rate by 9.9% for perampanel and 8.3% for lacosamide (P < .05). Among those with baseline hospitalizations, perampanel was associated with a 59.9% reduction in all-cause hospitalizations vs 48.6% for lacosamide (P < .05), and for epilepsy-related hospitalizations, a reduction of 65.0% vs 58.9%, respectively (P < .05). SIGNIFICANCE: Perampanel was associated with a significant reduction in one-year hospitalization risk.

GNAQ knockdown promotes bone metastasis through epithelial-mesenchymal transition in lung cancer cells.

AIMS: Bone metastasis ultimately occurs due to a complex multistep process, during which the interactions between cancer cells and bone microenvironment play important roles. Prior to colonization of the bone, cancer cells must succeed through a series of steps that will allow them to gain migratory and invasive properties; epithelial-to-mesenchymal transition (EMT) is known to be integral here. The aim of this study was to determine the effects of G protein subunit alpha Q (GNAQ) on the mechanisms underlying bone metastasis through EMT pathway. METHODS: A total of 80 tissue samples from patients who were surgically treated during January 2012 to December 2014 were used in the present study. Comparative gene analysis revealed that the GNAQ was more frequently altered in metastatic bone lesions than in primary tumour sites in lung cancer patients. We investigated the effects of GNAQ on cell proliferation, migration, EMT, and stem cell transformation using lung cancer cells with GNAQ-knockdown. A xenograft mouse model tested the effect of GNAQ using micro-CT analyses and histological analyses. RESULTS: GNAQ-knockdown showed down-regulation of tumour growth through mitogen-activated protein kinase (MAPK) signalling in lung cancer cells, but not increased apoptosis. We found that GNAQ-knockdown induced EMT and promoted invasiveness. GNAQ-knockdown cells injected into the bone marrow of murine tibia induced tumour growth and bone-to-lung metastasis, whereas it did not in control mice. Moreover, the knockdown of GNAQ enhanced cancer stem cell-like properties in lung cancer cells, which resulted in the development of resistance to chemotherapy. CONCLUSION: The present study reveals that the GNAQ-knockdown induced cancer stem cell-like properties. Cite this article: Bone Joint Res 2021;10(5):310-320.

Spin-Flip Density Functional Theory for the Redox Properties of Organic Photoredox Catalysts in Excited States.

Photoredox catalysts (PCs) have contributed to the advancement of organic chemistry by accelerating conventional reactions and enabling new pathways through the use of reactive electrons in excited states. With a number of successful applications, chemists continue to seek new promising organic PCs to achieve their objectives. Instead of labor-intensive manual experimentation, quantum chemical simulations could explore the enormous chemical space more efficiently. The reliability and accuracy of quantum chemical simulations have become important factors for material screening. We designed a theoretical protocol capable of predicting redox properties in excited states with high accuracy for a selected model system of dihydroquinoxalino[2,3-b]quinoxaline derivatives. Herein, three factors were considered as critical to achieving reliable predictions with accurate physics: the solvent medium effect on excited-state geometries, an adequate amount of Hartree-Fock exchange (HFX), and the consideration of double-excitation character in excited states. We determined that it is necessary to incorporate solvent medium during geometry optimizations to obtain planar excited-state structures that are consistent with the experimentally observed modest Stokes shift. While density functionals belonging to the generalized gradient approximation family perform well for the prediction of photoelectrochemical properties, an incorrect description of exciton boundedness (spontaneous dissociation of excitons or extremely weak boundedness) on small organic molecules was predicted. The inclusion of an adequate amount of Hartree-Fock exchange was suggested as one approach to obtain bound excitons, which is physically reasonable. The last consideration is the double-excitation character in S1 states. As revealed by the second-order algebraic diagrammatic construction theory, non-negligible double excitations exist in S1 states in our model systems. Time-dependent density functional theory (TDDFT) is blind to doubly excited states, and this motivated us to use spin-flip DFT (SF-DFT). We established a theoretical protocol that could provide highly accurate estimations of photophysical properties and ground-/excited-state redox properties, focusing on the three factors mentioned above. Geometry optimization with DFT and TDDFT employing the B3LYP functional (20% HFX) in solution and energy refinement by SF-DFT reproduced the experimental redox properties in the excited and ground states remarkably well with mean signed deviations (MSDs) of 0.01 and -0.15 V, respectively. This theoretical protocol is expected to contribute to the understanding of exciton behavior in organic PCs and to the efficient design of new promising PC candidates.

PTCH1 regulates anchorage-independent growth and bone invasion of non-small cell lung cancer cells.

Acquisition of metastatic potential by cancer cells is related to cancer stemness and anchorage-independent growth. The onset and progression of cancer are known to involve Hedgehog (HH) signaling that is activated by the binding of HH to the Patched 1 (PTCH1) receptor. However, the functions and mechanisms of action of PTCH1 in the context of bone metastasis remain to be elucidated. In this study, lentivirally-delivered shRNA was used to deplete PTCH1 levels, which resulted in the inhibition of spherical colony formation by the human non-small cell lung cancer (NSCLC) cell line; this suggested that PTCH1 promotes anchorage-independent growth. Concordantly, knockdown of PTCH1 resulted in significantly reduced migration and invasion of NSCLC cells; this was accompanied by the downregulation of MMP7 and SOX2. PTCH1 knockdown resulted in decreased bone destruction and osteoclastogenesis in a mouse bone metastasis model. These results indicate that PTCH1 may be an important regulator of bone invasion, and strongly suggest that knockdown of PTCH1 may decrease the anchorage-independent growth and metastatic potential of NSCLC.

Effect of GNAQ alteration on RANKL-induced osteoclastogenesis in human non-small-cell lung cancer.

AIMS: Receptor activator of nuclear factor-κB ligand (RANKL) is a key molecule that is expressed in bone stromal cells and is associated with metastasis and poor prognosis in many cancers. However, cancer cells that directly express RANKL have yet to be unveiled. The current study sought to evaluate how a single subunit of G protein, guanine nucleotide-binding protein G(q) subunit alpha (GNAQ), transforms cancer cells into RANKL-expressing cancer cells. METHODS: We investigated the specific role of GNAQ using GNAQ wild-type cell lines (non-small-cell lung cancer cell lines; A549 cell lines), GNAQ knockdown cell lines, and patient-derived cancer cells. We evaluated GNAQ, RANKL, macrophage colony-stimulating factor (M-CSF), nuclear transcription factor-κB (NF-κB), inhibitor of NF-κB (IκB), and protein kinase B (Akt) signalling in the GNAQ wild-type and the GNAQ-knockdown cells. Osteoclastogenesis was also evaluated in both cell lines. RESULTS: In the GNAQ-knockdown cells, RANKL expression was significantly upregulated (p < 0.001). The expression levels of M-CSF were also significantly increased in the GNAQ-knockdown cells compared with control cells (p < 0.001). GNAQ knockdown cells were highly sensitive to tumour necrosis factor alpha (TNF-α) and showed significant activation of the NF-κB pathway. The expression levels of RANKL were markedly increased in GNAQ mutant compared with GNAQ wild-type in patient-derived tumour tissues. CONCLUSION: The present study reveals that the alterations of GNAQ activate NF-κB pathway in cancers, which increase RANKL and M-CSF expression and induce osteoclastogenesis in cancers.Cite this article: Bone Joint Res. 2020;9(1):29-35.

The Humanistic and Economic Burden of Pediatric Focal Seizures in the United States.

OBJECTIVE: To better understand the humanistic and economic burden of focal seizures in children 2-12 years old. METHODS: We conducted a targeted literature review by searching MEDLINE for English-language publications reporting on children 2-12 years old with focal seizures published in the United States since 2008. RESULTS: Thirty-five publications were included. Incidence of focal seizures was 23.2 to 47.1 per 100,000 children per year; prevalence was 2.0 per 1,000 children, and ranged from 1.6 - 2.6 per 1,000 in patients of any age. Life expectancy was 47.3-61.8 years among children 3-12 years old. Patients took several antiepileptic drugs and experienced frequent seizures, sleep disorders, mood disorders, migraine, and seizure-related injuries (eg, bone fractures, sprains, open wounds). Children with focal seizures scored below average on cognitive assessments and up to 42%, 16%, and 19% had depression, anxiety, and attention-deficit disorder, respectively. Patients of any age had about 10 outpatient visits (2 epilepsy-related), 2 inpatient visits (less than 1 epilepsy-related), and 24 procedures (1 epilepsy-related) per year. Medication adherence was low: only half of pediatric patients maintained ≥90% adherence over 6 months. Annual total health care costs among patients of any age ranged from $18,369 - 38,549; first-year total health care costs for children were $19,883. CONCLUSIONS: Incidence and prevalence of focal seizures is high and the humanistic and economic burdens are significant. Future studies focused exclusively on children with focal seizures are needed to more precisely describe the burden. We also suggest further research and implementation of methods to improve medication adherence as an approach to lessen burden on these young patients.

Rapid determination of carbapenem resistance by low-cost colorimetric methods: Propidium Iodide and alamar blue staining.

Carbapenems are a class of ß-lactam antibiotics with a broad antimicrobial activity spectrum. Owing to their sturdy structures resistant to most ß-lactamases, they have been regarded as one of the last-resort antibiotics for combating multidrugresistant bacterial infections. However, the emergence of carbapenem resistance increases predominantly in nosocomial pathogens. To prevent spread of carbapenem resistance in early stages, it is imperative to develop rapid diagnostic tests that will substantially reduce the time and cost in determining carbapenem resistance. Thus, we devised a staining-based diagnostic method applicable to three different Gram-negative pathogens of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, all with the high potential to develop carbapenem resistance. Regardless of the resistance mechanisms presented by bacterial species and strains, double staining with propidium iodide (PI) and alamar blue (AB) identified resistant bacteria with an average sensitivity of 95.35%, 7 h after imipenem treatments in 343 clinical isolates. Among the three species tested, A. baumannii showed the highest diagnostic sensitivity of 98.46%. The PI and ABmediated staining method could be a promising diagnostic method with high-throughput efficacy and low cost.

Risk of hospitalization among patients with epilepsy using long versus short half-life adjunctive antiepileptic drugs.

INTRODUCTION: While antiepileptic drugs (AEDs) remain the primary treatment for epilepsy, many patients continue to have seizures. Uncontrolled seizures may be related to AED half-life, since short half-life (SHL) AEDs require more frequent dosing compared with the simplified regimens of long half-life (LHL) AEDs. Long half-life AEDs may also improve seizure control by extending missed dose forgiveness periods. The value of LHL AEDs may be assessed as reduced healthcare utilization. The study's objective was to examine the impact of adding an LHL versus SHL adjunctive AED on the risk of hospitalizations in patients with uncontrolled epilepsy. METHODS: This was a retrospective, longitudinal cohort study using the Symphony Health Solution Patient Integrated Dataverse. Patients ≥12 years old with uncontrolled epilepsy (≥2 medical claims ≥30 days apart) were identified during a study period (8/1/2012-7/31/2017). Patients were selected if they were subsequently initiated an adjunctive AED (excluding modified release formulations), and the prescription date served as the index. Patients were stratified into two mutually exclusive cohorts based on the index AED half-life (≤20 versus >20 h). Poisson regressions with robust error variances were performed for the relative risks (RRs) of all-cause, epilepsy-related, and injury-related hospitalizations. RESULTS: A total of 4984 patients were identified (2705 in the LHL and 2279 in the SHL cohort). Compared with those in the SHL cohort, patients in the LHL cohort were significantly younger [mean (SD, years): 43.9 (18.5) versus 49.2 (17.2), p < 0.001] and were less comorbid [mean (SD) of Charlson comorbidity index: 1.2 (1.8) versus 1.8 (2.2), p < 0.001]. In the one-year postindex date, adjusting for group differences, the risks of both all-cause and epilepsy-related hospitalizations were significantly lower in the LHL cohort than in the SHL cohort [all-cause: 0.84 (95% CI: 0.76-0.93), p = 0.0006; epilepsy-related: 0.83 (0.73-0.94), p = 0.0046].Injury-related hospitalizations did not differ between LHL and SHL cohorts. CONCLUSION: In patients with uncontrolled epilepsy who were initiated on an adjunctive AED, the choice of an LHL versus SHL was associated with significantly lower risks of all-cause and epilepsy-related hospitalizations.

Effect of AZD0530 on Cerebral Metabolic Decline in Alzheimer Disease: A Randomized Clinical Trial.

IMPORTANCE: Oligomeric amyloid-ß peptide binds to cellular prion protein on the neuronal cell surface, activating intracellular fyn kinase to mediate synaptotoxicity and tauopathy. AZD0530 is an investigational kinase inhibitor specific for the Src family, including fyn, that has been repurposed for the treatment of Alzheimer disease. OBJECTIVE: To determine whether AZD0530 treatment slows the decline in cerebral metabolic rate for glucose (CMRgl) and is safe and well tolerated. DESIGN, SETTING, AND PARTICIPANTS: This multicenter phase 2a randomized clinical trial enrolled participants between December 23, 2014, and November 30, 2016. Participants (n = 159) had mild Alzheimer dementia and positron emission tomography (PET) evidence of elevated levels of amyloid-ß peptide. Efficacy analyses of all primary and secondary outcomes were conducted in a modified intention-to-treat population. Final analyses were conducted from February 9, 2018, to July 25, 2018. INTERVENTIONS: AZD0530 (100 mg or 125 mg daily) vs placebo for 52 weeks. MAIN OUTCOMES AND MEASURES: Primary outcome was the reduction in relative CMRgl, as measured by 18F-fluorodeoxyglucose (18F-FDG) PET, at 52 weeks in an Alzheimer disease-associated prespecified statistical region of interest. Secondary end points included change in cognition, function, and other biomarkers. RESULTS: Among the 159 participants, 79 were randomized to receive AZD0530 and 80 to receive placebo. Of the 159 participants, 87 (54.7%) were male, with a mean (SD) age of 71.0 (7.7) years. Based on a week-2 plasma drug level (target = 180 ng/mL; 30nM free), 15 participants (19.2%) had their AZD0530 dose escalated from 100 mg to 125 mg. Mean plasma levels from weeks 13 to 52 were 220 ng/mL and 36nM free. More participants discontinued treatment with AZD0530 than with placebo (21 vs 11), most commonly because of adverse events. The most frequent adverse events were gastrointestinal disorders (primarily diarrhea), which occurred in 38 participants (48.1%) who received AZD0530 and in 23 (28.8%) who received placebo. In the primary outcome, the treatment groups did not differ in 52-week decline in relative CMRgl (mean difference: -0.006 units/y; 95% CI, -0.017 to 0.006; P = .34). The treatment groups also did not differ in the rate of change in Alzheimer's Disease Assessment Scale-Cognitive Subscale, Alzheimer's Disease Cooperative Study-Activities of Daily Living, Clinical Dementia Rating, Neuropsychiatric Inventory, or Mini-Mental State Examination scores. Secondary volumetric magnetic resonance imaging analyses revealed no treatment effect on total brain or ventricular volume but did show trends for slowing the reduction in hippocampal volume and entorhinal thickness. CONCLUSIONS AND RELEVANCE: Statistically significant effects of AZD0530 treatment were not found on relative CMRgl reduction in an Alzheimer disease-associated region of interest or on secondary clinical or biomarker measures. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02167256.

Activation of RIG-I-Mediated Antiviral Signaling Triggers Autophagy Through the MAVS-TRAF6-Beclin-1 Signaling Axis.

Autophagy has been implicated in innate immune responses against various intracellular pathogens. Recent studies have reported that autophagy can be triggered by pathogen recognizing sensors, including Toll-like receptors and cyclic guanosine monophosphate-adenosine monophosphate synthase, to participate in innate immunity. In the present study, we examined whether the RIG-I signaling pathway, which detects viral infections by recognizing viral RNA, triggers the autophagic process. The introduction of polyI:C into the cytoplasm, or Sendai virus infection, significantly induced autophagy in normal cells but not in RIG-I-deficient cells. PolyI:C transfection or Sendai virus infection induced autophagy in the cells lacking type-I interferon signaling. This demonstrated that the effect was not due to interferon signaling. RIG-I-mediated autophagy diminished by the deficiency of mitochondrial antiviral signaling protein (MAVS) or tumor necrosis factor receptor-associated factor (TRAF)6, showing that the RIG-I-MAVS-TRAF6 signaling axis was critical for RIG-I-mediated autophagy. We also found that Beclin-1 was translocated to the mitochondria, and it interacted with TRAF6 upon RIG-I activation. Furthermore, Beclin-1 underwent K63-polyubiquitination upon RIG-I activation, and the ubiquitination decreased in TRAF6-deficient cells. This suggests that the RIG-I-MAVS-TRAF6 axis induced K63-linked polyubiquitination of Beclin-1, which has been implicated in triggering autophagy. As deficient autophagy increases the type-I interferon response, the induction of autophagy by the RIG-I pathway might also contribute to preventing an excessive interferon response as a negative-feedback mechanism.

TP53-dependence on the effect of doxorubicin and Src inhibitor combination therapy.

The anticancer effects of Src kinase inhibitors are controversial. This study found an association between alterations in the TP53 gene and the synergy score for combination treatment with doxorubicin and an Src kinase inhibitor using human osteosarcoma cell lines (MG63 and U2OS) and human colon cancer cell line. Doxorubicin was found to activate signal transducer and activator of transcription 3 via Src kinase in cancer cells harboring alterations in TP53. A drug combination study using patient-derived cells confirmed that an Src kinase inhibitor synergizes with doxorubicin in cancer cells harboring alterations in TP53, while antagonizing its effect in cancer cells expressing wild-type TP53. Our findings suggest that genetic alterations in TP53 are a critical factor in determining the use of a combination treatment of doxorubicin and Src inhibitors.

Preclinical Evaluation of In Vitro and In Vivo Antiviral Activities of KCT-01, a New Herbal Formula against Hepatitis B Virus.

Hepatitis B virus (HBV) infectious diseases currently remain incurable due to limitations of conventional antivirals such as incapability of eradicating HBV DNA, prolonged use, drug resistance, and virological relapse. KCT-01, a 30% ethanol extract consisting of Artemisia capillaris, Sanguisorba officinalis, and Curcuma longa, was newly developed. The objective of this study was to investigate pharmacological activities of KCT-01 against HBV using HepG2.2.15 cells and a hydrodynamic injection model. KCT-01 significantly lowered antigen secretion, virion production, and pgRNA synthesis in HepG2.2.15 cells without affecting cell viability. KCT-01 administration also resulted in significant decrease of serum virion production, liver covalently closed circular (ccc) DNA levels, and mRNA synthesis of cytokines in the liver of mice injected with HBV DNA hydrodynamically. Interestingly, coadministration of KCT-01 with entecavir enhanced its in vitro and in vivo antiviral activities. Moreover, safety of KCT-01 was assured up to 5000 mg/kg in rats in both single and repeated-dose preclinical studies. Taken together, our findings demonstrate that KCT-01 is capable of suppressing HBV replication and inflammatory cytokine production in in vitro and in vivo models without showing toxicity, suggesting the potential of using KCT-01 alone or in combination with entecavir as antiviral agent.